This research project will study the topology of phospholipids and proteins in cell membranes. The topology will include asymmetric arrangement, clusters, and lipid-protein neighbors. Penetrating, non-penetrating and cross-linking covalent probes will be used for these studies. The following cell membranes will be studied: red blood cell, platelets, mitochondria, retinal rod outer segment disks, and bacteria. The factors which regulate or maintain phospholipid asymmetry will be studied in the red cell and platelet membrane. The extent of asymmetry of phosphatidylserine and phosphatidylethanolamine will be studied in normal humans and in patients with blood clotting disorders to see if there is a correlation between the accessability of the amino phospholipids on the outer membrane surface and blood clotting time. Various chemical probes will be used in sequence to test for heterogeneous phospholipid domains in cell membranes and to correlate these with the transport of inorganic cations and anions. The effect of drugs which act on cell membranes will be studied by covalent probes to see if these drugs modify phospholipid asymmetry and phospholipid clusters and phospholipid-protein interactions. The interaction of spectrin and rhodopsin with liposomes containing phosphatidylserine and phosphatidylethanolamine will serve as a model for lipid-protein interactions. Since we have synthesized a radioactive photoaffinity probe (NAP-propranolol) for the beta-adrenergic receptor, we will use the probe in conjunction with affinity chromatography to label, isolate and purify the beta-adrenergic receptor in the turkey red cell membrane. We will also continue our studies to elucidate the mechanism of the thyroxine (or triiodothyronine) stimulation of beta adrenergic receptors in heart ventricle membranes. Rat heart slices and isolated ventricle myocytes will be used for these studies. If patients are available, we shall measure the number and affinity of alpha and beta adrenergic receptors in lymphocytes from normal, hyperthyroid and hypothyroid patients and patients on propranolol therapy.